Real-time PCR analysis of prostein mRNA expression in matched samples of malignant and nonmalignant prostate tissues, in the PCa cell line LNCaP 1740, and the melanoma cell line 93.04A12.1. (A) To quantify the prostein mRNA expression in 15 paired cDNA samples of tumourous (black bars) and nontumourous (white bars) prostate tissues and in LNCaP 1740 and 93.04A12.1 cells a 240 bp fragment was amplified in a SYBR Green I-based LC assay. The transcript quantity was normalised to the expression level of HPRT. The results represent the means of two LC runs, bars indicate s.e. The ratio of prostein expression in the tumourous related to the transcript quantity in the corresponding nontumourous tissue sample (ratio T/NT) is given for each tissue pair. Patients' data were classified according to their tumour stage (pT) and within the groups according to the T/NT ratios. (B) The PCa cell line LNCaP 1740 was cultured for 48 h in the androgen-depleted medium (LNCaP a) or in the presence of 1 nM of the synthetic androgen R1881 (LNCaP b) and then used for RNA preparation and quantification of prostein transcripts. The results represent the means of two LC runs, bars indicate s.e.

In vitro generation of cytotoxic effector T cells specifically recognising the prostein-derived peptide 2004. Purified CD8+ T lymphocytes of two prostate cancer patients (donors 1 and 3) and one healthy donor (donor 2) were weekly stimulated by peptide-pulsed autologous DCs. After four stimulations T-cell cultures were tested for the activation of peptide-specific tumour-reactive CTLs. The stimulated T-cell cultures were added to 3 × 10³ peptide-pulsed T2 target cells well⁻¹ at an effector cell to target cell ratio of 20 : 1. Unloaded T2 cells and T2 cells pulsed with an irrelevant peptide from HIV reverse transcriptase served as controls. The results represent the mean values of triplicate determinations, bars indicate s.e.

HLA-A2 expression on the surface of the tumour cell lines used as target cells in the chromium release assays. The PCa cell line LNCaP 1740 (A), the melanoma cell line 93.04A12.1 (B) and the HLA-A2-negative PCa cell line PC-3 as control (C) were analysed by flow cytometry using the anti-HLA-A2 antibody MA2.1 as primary antibody and a PE-labelled goat anti-mouse IgG F(ab′)₂ fragment as secondary antibody. The solid line represents the IgG1 isotype control, the shaded peak represents the HLA-A2 staining.

Prostein-specific lysis and HLA-A^*0201-restricted recognition of LNCaP 1740 cells by the in vitro-generated cytotoxic effector cells. (A) After four rounds of stimulation activated CD8+ T cells from the three donors were cocultured with 3 × 10^{3 51}Cr-labeled LNCaP 1740, 93.04A12.1 or K562 tumour cells well⁻¹ at various effector cell (E) to target cell (T) ratios (3 : 1, 10 : 1, 30 : 1). After 4 h of incubation, chromium release was determined. (B) The inhibition of T-cell-mediated cytotoxicity against LNCaP 1740 cells was tested in the presence of the monoclonal anti-HLA-A2 antibody MA2.1 at an E : T ratio of 30 : 1. (C) Influence of androgen deprivation or supplementation on the prostein-specific lysis of LNCaP 1740 target cells. LNCaP 1740 cells were cultured for 24 h in medium containing charcoal-stipped FCS. Cells were grown for additional 48 h in the androgen-deprived medium or in the presence of 1 nM R1881 and then used as target cells in a chromium release assay at an E : T ratio of 30 : 1. All results represent the mean values of triplicate determinations, bars indicate s.e.